Metabolic and Morphologic Studies in Intraportal-Islet-Transplanted Rats Miriam A. Pipeleers-Marichal, M.D.,* Daniel G. Pipeleers, M.D.,* John Cutler, Paul E. Lacy, M.D., and David M. Kipnis, M.D., St. Louis
SUMMARY
The intraportal injection of 350 to 1,000 isolated islets into streptozotocin-diabetic rats immediately normalized (—24 hours) fasting plasma glucose and insulin levels. Polyuria, polydipsia, and hyperglucagonemia disappeared more gradually over a 2-to-12-week period—the time required for normalization varying with the severity of the diabetes and the number of islets transplanted. In long-term islet-transplanted rats (> five months), the hepatic insulin and glucagon reserves averaged 50 per cent and 25 per cent, respectively, of the corresponding normal pancreatic hormone content. Glucagon was increased slightly in the pancreas
of streptozotocin-diabetic rats and decreased considerably in transplanted animals. However, total pancreatic glucagon (i.e. pancreatic and hepatic reserves) in transplanted animals was the same as the pancreatic content of normal control rats, indicating the presence of feedback control mechanism(s) in the regulation of pancreatic glucagon reserves. Long-term transplanted islets demonstrated well-granulated A-, B-, and D-cell movement out of the vascular space and the formation of narrow intercellular spaces and junctional complexes with surrounding hepatocytes. DIABETES 25:1041-51, November, 1976.
Several studies have clearly demonstrated that islet transplantation is a biologically and technically feasible procedure. 1 " 4 However, the criteria used to assess the effectiveness of islet transplantation in reversing the diabetic state have generally been rather gross indices of severe insulin deficiency; namely, the fasting blood glucose level, the degree of polyuria, glucosuria and weight loss, and mortality. It is well recognized, however, that the long-term macro- and microvascular complications of diabetes account for the increased morbidity and mortality of both insulin-dependent and non-insulin-dependent diabetics. The pathophysiology of these chronic diabetic manifestations is
poorly understood, but it seems reasonable to suggest that they might be prevented if the metabolic disturbances observed in diabetes could be completely normalized. The present work has been undertaken to determine the extent to which islet transplantation can normalize the severely insulin-deficient rat and to determine whether the intraportal site of transplantation is associated with unique metabolic adaptations. This paper presents baseline biochemical, hormonal, and morphologic features of the intraportal-islettransplant model used in this series of studies.
From the Metabolism Division, Washington University School of Medicine, St. Louis, Missouri. •Present address: Vrije Universiteit Brussel, Kon. Elisabeth Stichting, J. Crocqlaan, 1, 1020 Brussels, Belgium. Address reprint requests to David M. Kipnis, M.D., Washington University School of Medicine, St. Louis, Mo. 63110. Accepted for publication June 8, 1976. NOVEMBER,
1976
MATERIALS AND METHODS Male adult rats of the inbred Lewis strain (Simonson, California) weighing 300-350 gm. were used exclusively. Rats were made diabetic by the intravenous injection of 75 mg. streptozotocin per kg. body weight (kindly donated by Dr. W. Dulin, Upjohn Co., Kalamazoo). Normal, diabetic, and transplanted rats were kept individually in metabolic cages and had free access to regular Purina rat chow and water. Urine 1041
INTRAPORTAL-ISLET-TRANSPLANTED RATS
volume and food and water intake were measured daily and total body weight was recorded weekly. Blood was drawn from a tail vein while the animals were under brief ether anesthesia; the two minutes of exposure to ether was found to have no influence on plasma glucose, insulin, and glucagon levels when compared with values obtained in unanesthetized rats from an indwelling jugular vein catheter inserted 12-18 hours before study. Blood samples were collected in chilled tubes containing heparin, EDTA, and Trasylol and centrifuged immediately at 4°. All plasma samples were stored at —20°. Plasma glucose was measured by the hexokinase assay5 and plasma insulin and glucagon were measured by a doubleantibody immunoassay procedure. 6 - 7 Insulin and glucagon content in the liver and pancreas of normal, diabetic, and long-term transplanted animals were determined by the extraction procedure of Melani et al. 8 Islets of Langerhans were isolated from adult Lewis rats by the collagenase method of Lacy and Kostianovsky.9 Following distention of the pancreas with Hank's solution (containing 0.2 per cent albumin, pH 7.4), the pancreas was excised, chopped into fine pieces, and digested for 10 minutes with collagenase (3- 5 mg./ml., Worthington). The islets were washed five times with Hank's solution, then handpicked and maintained at room temperature in 1 ml. Krebs-Ringer bicarbonate buffer (KRB, pH 7.4) containing 1 per cent albumin and 4.3 mM glucose. With this procedure, it is possible to obtain routinely about 600 islets from four rats. The insulin and glucagon content of the islets averaged 56.5 ± 9 . 8 ng./islet (1.42 ± 0.25 mU./islet) and 2.7 ± 0.2 ng./islet, respectively (n=15). No more than 120 minutes elapsed between decapitation of the first donor and the time of transplantation. Islets collected under these conditions displayed a normal insulin response to 16.5 mM glucose during 60 minutes' incubation at 37°—115 ± 10 fiV. insulin was released per islet exposed to 16.7 mM glucose as against 11 ± 3 /U-U. insulin/islet in the absence of glucose (n=5). Transplantation was performed four to 20 weeks after streptozotocin injection, by which time a "diabetic" steady state had been reached in terms of the degree of weight loss, polyuria, and polydipsia. The intraportal transplantation was carried out by suspending islets in 0.75 ml. sterile KRB (containing 1 per cent albumin) and injecting them through a siliconized fine-tip Pasteur pipet into a Bard infusion catheter—thin-wall needle inserted into the portal vein. This technique permitted the quantitative 1042
transfer of islets to the portal system. Sham-operated animals received KRB solution. Transplanted rats were sacrificed at various intervals after transplantation for microscopic studies of both pancreas and liver. Islet histology was examined by Gomori stain 1 0 for granulated B-cells, by Grimelius stain 11 for A cells, and by the Hellerstrom-Hellman stain 12 for D cells. Localization of transplanted islets in the liver for electron microscopy was accomplished as follows: Recipient rats were placed under pentobarbital anesthesia and perfusion of the liver through the portal vein was initiated in situ with 5 per cent glutaraldehyde (in phosphate buffer, pH 7.4). As rapidly as possible, the liver was dissected free of all attachments and mounted in an in-vitro perfusion system 13 and the perfusion continued for 15 minutes (15-20 ml./min.) at room temperature. The liver was then cut into 2-3-mm. slices that were kept in glutaraldehyde until embedded in paraffin. Serial sections were made from each liver slice and examined by Gomori stain. When an islet was identified, the portion of the islet remaining in the paraffin block was excised with a razor blade, deparaffinated, and prepared for electron microscopy. Light- and electronmicroscopic studies were also carried out on hepatic tissue contiguous with and distal from transplanted islets. RESULTS Baseline Values for Control and Diabetic Rats Baseline values for weight, food and water intake, and urine volume for three- and eight-month-old normal Lewis rats are shown in table 1. Approximately 25-30 per cent of animals injected with 75 mg. streptozotocin per kg. body weight died within the first two weeks. Two or three weeks after injection, a steady-state level of marked insulin deficiency was established, characterized by a body weight averaging 75 per cent of initial weight and marked polydypsia and polyuria (figure 1, table 1). Fasting plasma glucose levels in these animals exceeded 350 mg./lOO ml., plasma insulin was markedly depressed (4.1 ± 0.3 /i,U./ml.) and plasma glucagon significantly elevated (540 ± 33 jjg./ml.) (figure 2). Transplantation was carried out three weeks after streptozotocin injection to assure having a group of animals in a stabilized diabetic state. Metabolic Steady State After Islet Transplantation Water intake and urine volume decreased progressively after the intraportal injection of 350-1,000 isDIABETES, VOL. 2 5 , NO. 11
MIRIAM A. PIPELEERS-MARICHAL, M . D . , A N D ASSOCIATES
TABLE 1 Baseline values for normal, diabetic, and islet-transplant rats Parameter
Normal 3 months
Weight (gm.) Food intake (gm./24 hr.) H2O intake (ml724 hr.) Urine volume (ml./24 hr.) Glucose (mg./lOO ml.) Insulin (/zU./ml.) Insulin/glucose
318
Diabetic 3-8 months
8 months
± 5
442
237
± 10
Transplant 8 months
± 4
407
± 6
19.9 ± 0..9
13.1 ±
1.0
29.8
±
1.1
14.1 ± 0.7
33.8 ± 1.,2
33.1 ±
0.9
141.0
±
6.0
32.0 ± 1.0
23.3 ± 0. 2
24.7 ±
0.5
125.0
±
5.0
24.8 ± 0.8
103.3 ± 2. 2
98.1 ±
2.2
444.0
± 11.6
111.3 ± 2.6
22.4 ± 2..6
22.0 ±
2.3
4.1
0.22 ±
0.02
0.009 ±
0.22 ± 0.02
±
37.6 ± 3.0
0.3
0.34 ± 0.03
0.001
Values represent mean ± S.E.M. of 16 or more animals.
lets, reaching normal values two to 12 weeks after transplantation (figure 1). Body weight increased rapidly following transplantation and plateaued at a level (407 ± 6 gm.) that differed significantly (p 4 0 0 r STREPTOZ.j
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16
20
24
FIG. 1. Short-term and long-term effects of the intraportal transplantation of pancreatic islets in diabetic rats. All values represent the mean ± S.E.M. of at least 20 animals. NOVEMBER, 1976
DIABETIC
2
3
4
>4
NORMAL
MONTHS FIG. 2.
Fasting plasma glucose, insulin, and glucagon levels in normal, diabetic, and transplanted rats.
1043
INTRAPORTAL-ISLET-TRANSPLANTED RATS
At least two factors appear to influence the rate of remission of the diabetic state following transplantation. Increasing the number of islets transplanted from 350-450 to 500-600 resulted in a trend toward more rapid normalization (figure 3). When the number of islets transplanted was increased to 1,000, the normalization period was significantly and markedly shortened (p
SUMMARY
The intraportal injection of 350 to 1,000 isolated islets into streptozotocin-diabetic rats immediately normalized (—24 hours) fasting plasma glucose and insulin levels. Polyuria, polydipsia, and hyperglucagonemia disappeared more gradually over a 2-to-12-week period—the time required for normalization varying with the severity of the diabetes and the number of islets transplanted. In long-term islet-transplanted rats (> five months), the hepatic insulin and glucagon reserves averaged 50 per cent and 25 per cent, respectively, of the corresponding normal pancreatic hormone content. Glucagon was increased slightly in the pancreas
of streptozotocin-diabetic rats and decreased considerably in transplanted animals. However, total pancreatic glucagon (i.e. pancreatic and hepatic reserves) in transplanted animals was the same as the pancreatic content of normal control rats, indicating the presence of feedback control mechanism(s) in the regulation of pancreatic glucagon reserves. Long-term transplanted islets demonstrated well-granulated A-, B-, and D-cell movement out of the vascular space and the formation of narrow intercellular spaces and junctional complexes with surrounding hepatocytes. DIABETES 25:1041-51, November, 1976.
Several studies have clearly demonstrated that islet transplantation is a biologically and technically feasible procedure. 1 " 4 However, the criteria used to assess the effectiveness of islet transplantation in reversing the diabetic state have generally been rather gross indices of severe insulin deficiency; namely, the fasting blood glucose level, the degree of polyuria, glucosuria and weight loss, and mortality. It is well recognized, however, that the long-term macro- and microvascular complications of diabetes account for the increased morbidity and mortality of both insulin-dependent and non-insulin-dependent diabetics. The pathophysiology of these chronic diabetic manifestations is
poorly understood, but it seems reasonable to suggest that they might be prevented if the metabolic disturbances observed in diabetes could be completely normalized. The present work has been undertaken to determine the extent to which islet transplantation can normalize the severely insulin-deficient rat and to determine whether the intraportal site of transplantation is associated with unique metabolic adaptations. This paper presents baseline biochemical, hormonal, and morphologic features of the intraportal-islettransplant model used in this series of studies.
From the Metabolism Division, Washington University School of Medicine, St. Louis, Missouri. •Present address: Vrije Universiteit Brussel, Kon. Elisabeth Stichting, J. Crocqlaan, 1, 1020 Brussels, Belgium. Address reprint requests to David M. Kipnis, M.D., Washington University School of Medicine, St. Louis, Mo. 63110. Accepted for publication June 8, 1976. NOVEMBER,
1976
MATERIALS AND METHODS Male adult rats of the inbred Lewis strain (Simonson, California) weighing 300-350 gm. were used exclusively. Rats were made diabetic by the intravenous injection of 75 mg. streptozotocin per kg. body weight (kindly donated by Dr. W. Dulin, Upjohn Co., Kalamazoo). Normal, diabetic, and transplanted rats were kept individually in metabolic cages and had free access to regular Purina rat chow and water. Urine 1041
INTRAPORTAL-ISLET-TRANSPLANTED RATS
volume and food and water intake were measured daily and total body weight was recorded weekly. Blood was drawn from a tail vein while the animals were under brief ether anesthesia; the two minutes of exposure to ether was found to have no influence on plasma glucose, insulin, and glucagon levels when compared with values obtained in unanesthetized rats from an indwelling jugular vein catheter inserted 12-18 hours before study. Blood samples were collected in chilled tubes containing heparin, EDTA, and Trasylol and centrifuged immediately at 4°. All plasma samples were stored at —20°. Plasma glucose was measured by the hexokinase assay5 and plasma insulin and glucagon were measured by a doubleantibody immunoassay procedure. 6 - 7 Insulin and glucagon content in the liver and pancreas of normal, diabetic, and long-term transplanted animals were determined by the extraction procedure of Melani et al. 8 Islets of Langerhans were isolated from adult Lewis rats by the collagenase method of Lacy and Kostianovsky.9 Following distention of the pancreas with Hank's solution (containing 0.2 per cent albumin, pH 7.4), the pancreas was excised, chopped into fine pieces, and digested for 10 minutes with collagenase (3- 5 mg./ml., Worthington). The islets were washed five times with Hank's solution, then handpicked and maintained at room temperature in 1 ml. Krebs-Ringer bicarbonate buffer (KRB, pH 7.4) containing 1 per cent albumin and 4.3 mM glucose. With this procedure, it is possible to obtain routinely about 600 islets from four rats. The insulin and glucagon content of the islets averaged 56.5 ± 9 . 8 ng./islet (1.42 ± 0.25 mU./islet) and 2.7 ± 0.2 ng./islet, respectively (n=15). No more than 120 minutes elapsed between decapitation of the first donor and the time of transplantation. Islets collected under these conditions displayed a normal insulin response to 16.5 mM glucose during 60 minutes' incubation at 37°—115 ± 10 fiV. insulin was released per islet exposed to 16.7 mM glucose as against 11 ± 3 /U-U. insulin/islet in the absence of glucose (n=5). Transplantation was performed four to 20 weeks after streptozotocin injection, by which time a "diabetic" steady state had been reached in terms of the degree of weight loss, polyuria, and polydipsia. The intraportal transplantation was carried out by suspending islets in 0.75 ml. sterile KRB (containing 1 per cent albumin) and injecting them through a siliconized fine-tip Pasteur pipet into a Bard infusion catheter—thin-wall needle inserted into the portal vein. This technique permitted the quantitative 1042
transfer of islets to the portal system. Sham-operated animals received KRB solution. Transplanted rats were sacrificed at various intervals after transplantation for microscopic studies of both pancreas and liver. Islet histology was examined by Gomori stain 1 0 for granulated B-cells, by Grimelius stain 11 for A cells, and by the Hellerstrom-Hellman stain 12 for D cells. Localization of transplanted islets in the liver for electron microscopy was accomplished as follows: Recipient rats were placed under pentobarbital anesthesia and perfusion of the liver through the portal vein was initiated in situ with 5 per cent glutaraldehyde (in phosphate buffer, pH 7.4). As rapidly as possible, the liver was dissected free of all attachments and mounted in an in-vitro perfusion system 13 and the perfusion continued for 15 minutes (15-20 ml./min.) at room temperature. The liver was then cut into 2-3-mm. slices that were kept in glutaraldehyde until embedded in paraffin. Serial sections were made from each liver slice and examined by Gomori stain. When an islet was identified, the portion of the islet remaining in the paraffin block was excised with a razor blade, deparaffinated, and prepared for electron microscopy. Light- and electronmicroscopic studies were also carried out on hepatic tissue contiguous with and distal from transplanted islets. RESULTS Baseline Values for Control and Diabetic Rats Baseline values for weight, food and water intake, and urine volume for three- and eight-month-old normal Lewis rats are shown in table 1. Approximately 25-30 per cent of animals injected with 75 mg. streptozotocin per kg. body weight died within the first two weeks. Two or three weeks after injection, a steady-state level of marked insulin deficiency was established, characterized by a body weight averaging 75 per cent of initial weight and marked polydypsia and polyuria (figure 1, table 1). Fasting plasma glucose levels in these animals exceeded 350 mg./lOO ml., plasma insulin was markedly depressed (4.1 ± 0.3 /i,U./ml.) and plasma glucagon significantly elevated (540 ± 33 jjg./ml.) (figure 2). Transplantation was carried out three weeks after streptozotocin injection to assure having a group of animals in a stabilized diabetic state. Metabolic Steady State After Islet Transplantation Water intake and urine volume decreased progressively after the intraportal injection of 350-1,000 isDIABETES, VOL. 2 5 , NO. 11
MIRIAM A. PIPELEERS-MARICHAL, M . D . , A N D ASSOCIATES
TABLE 1 Baseline values for normal, diabetic, and islet-transplant rats Parameter
Normal 3 months
Weight (gm.) Food intake (gm./24 hr.) H2O intake (ml724 hr.) Urine volume (ml./24 hr.) Glucose (mg./lOO ml.) Insulin (/zU./ml.) Insulin/glucose
318
Diabetic 3-8 months
8 months
± 5
442
237
± 10
Transplant 8 months
± 4
407
± 6
19.9 ± 0..9
13.1 ±
1.0
29.8
±
1.1
14.1 ± 0.7
33.8 ± 1.,2
33.1 ±
0.9
141.0
±
6.0
32.0 ± 1.0
23.3 ± 0. 2
24.7 ±
0.5
125.0
±
5.0
24.8 ± 0.8
103.3 ± 2. 2
98.1 ±
2.2
444.0
± 11.6
111.3 ± 2.6
22.4 ± 2..6
22.0 ±
2.3
4.1
0.22 ±
0.02
0.009 ±
0.22 ± 0.02
±
37.6 ± 3.0
0.3
0.34 ± 0.03
0.001
Values represent mean ± S.E.M. of 16 or more animals.
lets, reaching normal values two to 12 weeks after transplantation (figure 1). Body weight increased rapidly following transplantation and plateaued at a level (407 ± 6 gm.) that differed significantly (p 4 0 0 r STREPTOZ.j
200 £
•»
o
*
100
Transplant
400
LU
8 200
0
nnnn n n
o
160 ULJ
^ 120 3 a.
z £ 80 CM
X
1
40
Z 3
40
1
JL
20
0
0 .£ 600r
130
o
Q.
90
z 400 o o y 200
50 10
1 1
6 -4
0
4
8 12 WEEKS
16
20
24
FIG. 1. Short-term and long-term effects of the intraportal transplantation of pancreatic islets in diabetic rats. All values represent the mean ± S.E.M. of at least 20 animals. NOVEMBER, 1976
DIABETIC
2
3
4
>4
NORMAL
MONTHS FIG. 2.
Fasting plasma glucose, insulin, and glucagon levels in normal, diabetic, and transplanted rats.
1043
INTRAPORTAL-ISLET-TRANSPLANTED RATS
At least two factors appear to influence the rate of remission of the diabetic state following transplantation. Increasing the number of islets transplanted from 350-450 to 500-600 resulted in a trend toward more rapid normalization (figure 3). When the number of islets transplanted was increased to 1,000, the normalization period was significantly and markedly shortened (p
